The rewiring of transcriptional circuits is a major source of evolutionary novelty. This proposal seeks to determine the detailed molecular mechanisms that underlie transcriptional rewiring using unicellular yeasts as a model system. The strategy is based on direct experimentation in three yeasts2Saccharomyces cerevisiae, Kluyveromyces lactis, and Candida albicans2using gene knockout collections, genome wide transcriptional profiling, chromatic immunoprecipitation, and phylogentic comparisons. Circuit comparisons among these three yeasts reveal specific examples of transcriptional rewiring and provide hypotheses for the molecular mechanisms by which the wiring changes occurred. Specific hypotheses will be tested and refined by phylogenetic comparisons of the over 40 sequenced ascomycete genomes and by additional experiments, including the 3resurrection4 and study of ancient proteins in modern yeasts. Prior and ongoing work on the evolution of the well-characterized yeast mating-type circuitry will serve as a guide; additional studies will include the evolution of regulatory circuits controlling metabolic pathways.